摘要
为揭示烟草糖基转移酶的活性及生物学功能,从烟草栽培品种中克隆了烟草糖基转移酶基因NtGT5a,基于GenBank的烟草糖基转移酶基因NtGT5a的核苷酸序列(GenBank登录号AB176523),设计并合成特异性引物,以红花大金元叶片总RNA为模板,通过RT-PCR方法获得了烟草糖基转移酶基因NtGT5a的cDNA片段。序列分析表明,该基因编码区为1458 bp,编码485个氨基酸残基,推测的蛋白分子量为54.21 kDa,理论等电点为5.41。通过构建重组表达载体pCold-SUMO-NtGT5a,并转化大肠杆菌BL21(DE3),在15Ⅲ下经0.2 mmol/L IPTG诱导22 h表达产生了SUMO-NtGT5a重组蛋白。重组蛋白部分以包涵体形式存在,部分以可溶性蛋白形式存在。将重组蛋白的可溶性组分经过Ni-NTA柱层析纯化和凝胶过滤层析,用SUMO蛋白酶切除SUMO标签,再用镍柱纯化获得了高纯度的NtGT5重组蛋白。
For investigating the activity and biological functions of glucosyltransferase in Nicotiana tabacum, gene NtGT5a was cloned from tobacco cultivars. By referring to the nucleotide sequence of NtGT5a of Nicotiana tabacum published on GenBank (GenBank accession number AB176523) , specific primers were designed and synthesized. The cDNA fragment of NtGT5a was obtained with RT-PCR by using the total RNA from leaves of cv. Honghuadajinyuan as template. Sequence analysis showed that the gene contained a full coding region of 1458 bp, encoded 485 amino acid residues with the estimated protein molecular mass of 54.21 kDa and the theoretical isoelectric point of 5.41. The coding sequence of NtGT5a gene was cloned into the prokaryotic expression vector pCold-SUMO, and this construct was expressed in E. coli BL21 (DE3). The recombinant protein SUMO-NtGT5a was generated via 0.2 mmol/L 1PTG (isopropyl-^-D-thiogalactopyranoside) induction at 15 ~C for 22 hours. Part of the recombinant protein was presented in the form of soluble protein and the other in the form of inclusion body. The supernatant containing the soluble target protein was purified by Ni-NTA column chromatography and gel filtration chromatography. The fusion tag was further removed by the SUMO protease, and the target recombinant protein NtGT5 of high purity was obtained by repurification with Ni-NTA column chromatography.
出处
《烟草科技》
EI
CAS
北大核心
2014年第7期69-74,84,共7页
Tobacco Science & Technology
基金
贵州省科技厅农业攻关项目"烤烟种质资源鉴定与创新及新品种选育研究"(黔科合NY字[2011]3047号)
中国烟草总公司重点项目"烤烟育种新技术与新一代烤烟新品种选育研究"(中烟办[2010]221号)
贵州省优秀青年科技人才培养对象专项资金"贵州主推烟草品种基因组结构研究"(黔科合人字[2013]02号)
关键词
烟草
糖基转移酶
基因
蛋白酶
Nicotiana tabacum
Glucosyltransferase
Gene
Protease